Centromere and Kinetochore Proteins

Centromeres are the chromosomal regions responsible for poleward movement at meiosis and mitosis, and are essential for the faithful segregation of
genetic information. Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Centromeric proteins include chromosomal passenger complex. It's name stems from the observation that these proteins colocalise on condensing chromosomes during prophase, and are carried along to centromeres and to the equator of the mitotic spindle during metaphase. After metaphase, the components re-localise to the midzone and midbody of the spindle, where they remain until the completion of cytokinesis. For information on the structure and constituents of centromeres and kinetochores, see Organization of the animal kinetochore in The dynamic kinetochore-microtubule interface.

It is well-established that centromeric regions and their function are influenced by epigenetic mechanisms to maintain their identity throughout cell and organismal generations. The histone variant CENP-A has been singled out as a key player in determining centromeres in most organisms studied so far. However, diversity and differences within centromeres suggest that additional mechanisms also play a role in centromere determination. This study provides evidence that the SAT III transcripts from a highly repetitive region of the X chromosome of D. melanogaster are important to maintain correct centromeric function, and therefore normal chromosome segregation. SAT III RNA depletion causes severe chromosome segregation defects and a partial loss of essential kinetochore components that mediate the interaction with the mitotic spindle. Furthermore, SAT III RNA interacts with the inner kinetochore protein CENP-C. A model is proposed where SAT III RNA binds to CENP-C, which in turn is required to recruit or stabilize CENP-C and possibly CENP-C–interacting factors such as CENP-A at centromeres. When SAT III RNA is absent, the association of CENP-C with centromeres is destabilized or inhibited, which impairs the association of other proteins that are dependent on CENP-C for their centromeric localization. Reciprocally, in the absence of CENP-C, SAT III is absent from centromeres, which suggests an interdependence of SAT III RNA and CENP-C. CENP-C, together with CENP-A and CAL1, forms a platform for binding of KMN proteins (named for the Knl1 complex, the Mis12 complex and the Ndc80 complex), which are required for the attachment of chromosomes to the mitotic spindle. Therefore, it is proposed that as a consequence of the SAT III depletion, chromosome missegregation is caused by the destabilization of centromeric chromatin and therefore kinetochore formation during mitosis (Rosic, 2014).

SAT III is transcribed in D. melanogaster embryos and adult flies (Usakin, 2007; Salvany, 2009). Long centromeric transcripts have been identified in other species as well. Even though long SAT III transcripts are predominantly detected, the existence of smaller transcripts cannot be excluded, as rapid centromeric transcript turnover has been described previously. In maize, centromeric transcripts remain bound to the kinetochore after transcription, and are thought to participate in stabilization of centromeric chromatin. Maize RNA binds to centromeric protein CENP-C transiently, and promotes its binding to DNA. Therefore, noncoding RNA may play a role similar to a protein chaperone. Once CENP-C is localized to centromeres, DNA binding is facilitated with the help from RNA to stabilize its position. During interphase, SAT III RNA localizes to the nucleus, and forms a cluster in proximity to sites of centromeric clusters, perhaps at its transcription site. During mitosis, SAT III RNA is present at centromeric regions. It is suggested that satellite transcripts function in stabilizing the centromeric positioning of CENP-C, thereby facilitating the building of kinetochore structures, and in turn require CENP-C to localize to centromeres. This mechanism may be evolutionarily conserved, as CENP-C has been described to bind RNA from centromeric repeats in maize. In addition to SAT III RNA present at centromeres, some SAT III RNA is also detectable at pericentromeres of mitotic chromosomes and is non-chromatin-associated. SAT III RNA that is present at pericentromeres might also contribute to overall kinetochore structure, and signals distant from chromatin might represent distinct ribonucleoprotein particles. However, additional work is required to address these questions (Rosic, 2014).

Depletion of SAT III RNA in S2 cells caused severe mitotic defects, which indicates that SAT III RNA is crucial for cell division. The same phenotype was observed in vivo in D. melanogaster embryos. Importantly, flies carrying an X-Y translocation chromosome that has lost most of its SAT III DNA block do not transcribe any significant amount of SAT III RNA, and display segregation defects in early embryos similar to what what was described for S2 cells and SAT III LNA gapmer-injected embryos. Most of the Zhr1 flies are viable and fertile despite the segregation defects in early embryos. It is therefore suggeste that SAT III RNA function is only one part of a larger safeguard mechanism required for accurate chromosome segregation during mitosis.
It has been shown that Zhr1 male flies rescue the female hybrid lethality in crosses between D. simulans females and D. melanogaster males. One of their hypotheses was that RNA originating from SAT III might be the cause of hybrid lethality in F1 daughters originating from these crosses. This study shows that Zhr1 flies do not have any SAT III transcripts, which indicates a possible incompatibility of SAT III RNA from wild-type D. melanogaster flies with either transcripts or the sequence of the X chromosome of D. simulans. However, this and other possibilities need to be tested in the future (Rosic, 2014).

A previous study showed that transcription of SAT III depends on the homeobox-containing transcription factor Hth, and mutations of hth lead to abnormal distribution of CENP-A (Salvany, 2009). Similarly, inhibition of transcription during mitosis resulted in a decreased level of centromeric α-satellite transcripts in human cells, which in turn resulted in lagging chromosomes and a reduction of CENP-C. Inhibition of transcription or mutations of transcription factors may, however, cause pleiotropic effects in cells; together with the results presented from a direct depletion of SAT III transcripts, this study concludes that the SAT III RNA directly influences centromere function and that satellite transcripts may have a conserved function in kinetochore formation (Rosic, 2014).

The inability of chromosomes to segregate properly in the absence of SAT III RNA is not restricted to chromosome X, the origin of SAT III transcripts. This indicates a trans-acting mechanism, as seen in dosage compensation and proposed for maize centromeric RNA. It has been suggested that each centromere is capable of producing RNA. Indeed, in D. melanogaster, active centromeric transcription by RNA polymerase II was observed on all chromosomes. This indicates that centromeric RNAs might have redundant functions, similar to what is described for the dosage compensation complex in Drosophila. Here, roX1 and roX2 RNA are required for spreading of the compensasome to the entire X chromosome. These two RNAs are redundant in their function, even though they have little sequence similarity. The presence of redundant RNAs may also explain why the majority of chromosomes usually segregate correctly upon SAT III RNA depletion, and why only some chromosomes are lagging (Rosic, 2014).

This study shows that SAT III RNA function is independent of heterochromatin formation. In support of this, Usakin (2007) reported that many D. melanogaster pericentromeric transcripts participate in heterochromatin formation, but SAT III transcripts were not among the RNAs that had an effect on the formation of centromeric heterochromatin. The observed heterochromatin defects in hth mutant embryos (Salvany, 2009) are, therefore, possibly caused by additional effects of depleting this transcription factor. Pericentromeric heterochromatin is required for sister chromatid cohesion and bipolar orientation during mitosis. However, the levels of cohesion proteins, as well as the heterochromatin markers HP1 and H3 lysine 9 methylation, are unaffected in SAT III–depleted cells. It is therefore concluded that the observed chromosome segregation defects after SAT III depletion are unlikely to be caused by a loss of sister chromatid cohesion or heterochromatin integrity (Rosic, 2014).

Levels of centromeric and kinetochore proteins were significantly reduced on mitotic chromosomes that failed to segregate properly in the absence of SAT III RNA, which implies a role of SAT III RNA in providing a competent centromere environment. Additionally, reducing the levels of CENP-C by RNAi caused a complete loss of SAT III from centromeres, which suggests that CENP-C and SAT III RNA are mutually dependent on each other for their centromeric localization. Because loading of CENP-C and CENP-A is mutually dependent as well, both proteins are reduced in the absence of SAT III, as expected. Spc105 is an essential component of Drosophila kinetochores; its localization is interdependent with MIS12 complex localization and required for localization of the NDC80 subcomplex, which directly binds microtubules. Hence, reduction of Spc105 protein at centromeres leads to severe defects in constructing a functional kinetochore, and provides an explanation for failures in chromosome segregation in the absence of SAT III RNA. Finally, SNAP tag experiments showed that loading of newly synthesized CENP-A and CENP-C proteins is also affected by the loss of SAT III, which suggests that SAT III plays an integral role in establishing and stabilizing centromeric chromatin. In conclusion, SAT III RNA was identified as an epigenetic factor involved in centromere regulation and function through interaction with the centromeric protein CENP-C, which suggests a vital and evolutionarily conserved role of noncoding RNAs in centromere determination and chromosome segregation (Rosic, 2014).

Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. Previous work has shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. This report shows that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. These results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis (Carmena, 2014).

Unlike other organisms that have evolved distinct H2A variants for different functions, Drosophila melanogaster has just one variant which is capable of filling many roles. This protein, H2A.V, combines the
features of the conserved variants H2A.Z and H2A.X in transcriptional control/heterochromatin assembly and DNA damage response, respectively. This study shows that mutations in the gene encoding H2A.V affect chromatin compaction and perturb chromosome segregation in Drosophila mitotic cells. A microtubule (MT) regrowth assay after cold exposure revealed that loss of H2A.V impaired the formation of kinetochore-driven (k) fibers, which could account for defects in chromosome segregation. All defects were rescued by a transgene encoding H2A.V that lacked the H2A.x function in the DNA damage response, suggesting that the H2A.Z (but not H2A.X) functionality of H2A.V was required for chromosome segregation. Loss of H2A.V weakened HP1
localization, specifically at the pericentric heterochromatin of
metaphase chromosomes. Interestingly, loss of HP1 yielded not only telomeric fusions but also mitotic defects similar to those seen in
H2A.V null mutants, suggesting a role for HP1 in chromosome
segregation. H2A.V precipitated HP1 from larval brain extracts
indicating that both proteins were part of the same complex.
Moreover, the overexpression of HP1 rescued chromosome
missegregation and defects in the kinetochore-driven k-fiber
regrowth of H2A.V mutants indicating that both phenotypes were
influenced by unbalanced levels of HP1. Collectively, these results suggest that H2A.V and HP1 work in concert to ensure kinetochore-driven MT growth (Verní, 2015).

This study provides compelling evidence that H2A.V, the Drosophila histone H2A variant, plays an important and unanticipated role during Drosophila mitosis. The cytological characterization of H2A.V810 mutant larval brain chromosomes revealed that loss of H2A.V has an impact on chromosome organization and cell proliferation, which is consistent with previous results on a role of this histone variant in chromatin remodeling and heterochromatin organization. This study also demonstrates that a significant proportion of H2A.V mutant cells fails to complete mitosis and contains chromosomes that remain scattered across the spindle (pseudo anaphase or PA) due to failed metaphase plate alignment. Similar effects have been previously described in Drosophila S2 cells depleted by RNAi of either kinetochore proteins, augmin components or splicing factors. However unlike those S2 interfered cells, which exhibit PAs with long spindles, H2A.V810 mutant cells have PA (premature- or pseudo-anaphase) spindles that appear similar to wild type anaphases. The reason why H2A.V mutant cells are not elongated is unclear, but it may depend on the different cellular systems employed in the different studies. Intriguingly, the presence of PAs in H2A.V810 mutants indicates for the first time that Drosophila H2A.V is also necessary for chromosome segregation growth (Verní, 2015).

Interestingly, the results from both Dgt6 immunolocalization and spindle microtubule re-growth assay following cold-induced MT depolymerization in mitotic neuroblasts reveal that H2A.V might be involved in the organization of kinetochore-driven, k-fibers microtubule bundles that attach sister kinetochores to spindle poles. However, it is believed that defects in the organization of k-fibers are not a consequence of the reduction of Dgt6. Recent studies demonstrated that Wac, a newly discovered component of Augmin complex, is required for spindle formation in S2 cells but is dispensable for somatic mitosis. In fact, a wac deletion mutant was viable and displayed only weak defects in brain cell divisions, suggesting that the components of Augmin complex (including Dgt6) might have non essential roles in spindle assembly growth (Verní, 2015).

It has been previously reported that defective k-fiber formation and elongation disrupt chromosome segregation and spindle formation in Drosophila cells. The results, which are in line with this finding, indicate that a specific chromatin organization is also necessary to ensure a proper spindle assembly. It is speculated that the observed PAs are a result of improper organization of k-fibers, and that PAs fail to complete mitosis, thus reducing in part the frequency of anaphases in H2A.V810 mutants. It is also plausible that persistent chromosome misalignment leads to a mitotic arrest of these cells, which in turn could explain the presence of H2A.V810 mutant cells with overcondensed chromosomes. However, while in a previous study, the presence of PAs was always associated to a strong increase of mitotic index (MI), the current mutants the MI did not change. One explanation is that the reduction of anaphase frequency in H2A.V810 mutants (20%) is not as dramatic as that reported for Dgt6-depleted S2 cells (50%) and therefore it unlikely affects mitotic progression. An alternative explanation is that loss of H2A.V might affect the regulation of G2-M and/or M-A cell cycle checkpoints thus preventing a metaphase arrest. Further investigations are required to verify this hypothesis. It is worth noting that, although a role for H2A.Z in chromosome segregation has been previously documented in human and yeast cells, the current data provide the first evidence of an potential involvement of H2A.Z in the organization of k-fibers growth (Verní, 2015).

This study also provides unanticipated molecular evidence that H2A.V interacts directly or indirectly with HP1, confirming that both proteins are part of same complex. It is intriguing that the H2A.V-HP1 interaction depends on the HP1 CD domain, which also binds H3K9me2/3 and mediates heterochromatin formation. This supports the existence of a cascade of events that requires the recruitment of H2A.V and different histone modifications for the establishment of heterochromatin. Yet, the reason why depletion of H2A.V causes a direct loss of HP1 and particularly during mitosis is unclear. Nonetheless, as HP1 overexpression in H2A.V mutant cells prevents PA formation, it is speculated that a H2A.V-dependent stabilization/localization of HP1 at centromeric region is essential to ensure proper chromosomal behavior growth (Verní, 2015).

Previous studies have shown that H2A.Z alters the nucleosomal surface, thus enabling preferential binding of HP1a to condensed higher chromatin structures 44RIDcit0044. It is conceivable then that the H2A.V-HP1 interaction is favored by the condensation of pericentric chromatin fiber in metaphase. Alternatively, these interactions may be encouraged by metaphase-specific posttranslational modifications of H2A.V, HP1 or other interacting proteins. Indeed, it has been proposed the mechanism underpinning HP1 recruitment on mitotic chromosomes might be different from that in interphase. Still, little is known about the factors required for specific localization of HP1 at mitotic centromeres save for a few discoveries. Human HP1α binding to INCENP, for instance, has been demonstrated as necessary for HP1α targeting to mitotic centromeres. It is believed that H2A.V may play a similar role in mediating HP1 binding, but how this takes place remains to be seen growth (Verní, 2015).

This functional characterization of H2A.V has also unveiled the role of Drosophila HP1a in the assembly of the mitotic spindle. The results indicate that the loss of HP1 yields defects in the kinetochore-driven k-fiber organization, which can in turn compromise chromosome segregation thus generating PAs. Past studies have shown that HP1a contributes to chromosome segregation and centromere stability in a variety of organisms including mammals, but the mechanism is still not completely understood. HP1 is known to interact with components of the centromere and the kinetochore complex, providing targets to begin understanding how downregulation or mislocalization of HP1 result in mitotic defects. It has also been reported that in contrast to Swi6 in S. pombe, the correct localization of HP1 is not required for the recruitment of cohesins to centromeric regions in mammals. Yet, HP1a seems to help in protecting cohesins from degradation by recruiting the Shugoshin protein. This study has highlighted an additional function of HP1 during chromosome segregation, one which depends on interaction with H2A.V and is required to regulate k-fiber organization. These results thus provide further evidence of a functional versatility of HP1 that is likely conserved also in mammals growth (Verní, 2015).

In oocytes, where centrosomes are absent, the chromosomes direct the assembly of a bipolar spindle. Interactions between chromosomes and microtubules are essential for both spindle formation and chromosome segregation. This study examined oocytes lacking two kinetochore proteins, NDC80 and SPC105R, and a centromere-associated motor protein, CENP-E, to characterize the impact of kinetochore-microtubule attachments on spindle assembly and chromosome segregation in Drosophila oocytes. The initiation of spindle assembly was shown to result from chromosome-microtubule interactions that are kinetochore-independent. Stabilization of the spindle, however, depends on both central spindle and kinetochore components. This stabilization coincides with changes in kinetochore-microtubule attachments and bi-orientation of homologs. It is proposed that the bi-orientation process begins with the kinetochores moving laterally along central spindle microtubules towards their minus ends. This movement depends on SPC105R, can occur in the absence of NDC80, and is antagonized by plus-end directed forces from the CENP-E motor. End-on kinetochore-microtubule attachments that depend on NDC80 are required to stabilize bi-orientation of homologs. A surprising finding was that SPC105R but not NDC80 is required for co-orientation of sister centromeres at meiosis I. Together, these results demonstrate that, in oocytes, kinetochore-dependent and -independent chromosome-microtubule attachments work together to promote the accurate segregation of chromosomes (Radford, 2015).

It is well established that oocyte spindle assembly in many organisms occurs in the absence of centrosomes. Instead, chromatin-based mechanisms play an important role in spindle assembly. The interactions between chromosomes and microtubules are paramount in oocytes, necessary for both the assembly of the spindle and the forces required for chromosome segregation. Less well understood, however, is the nature of the functional connections between chromosomes and microtubules in these cells. The role of the kinetochores, the primary site of interaction between chromosomes and microtubules, is poorly understood in acentrosomal systems. For example, spindles will assemble and chromatin will move without kinetochores in both Caenorhabditis elegans and mouse oocytes. In addition, both C. elegans and mouse oocytes experience a prolonged period during which chromosomes have aligned but end-on kinetochore-microtubule attachments have not formed. Previously shown that the central spindle, composed of antiparallel microtubules that assemble adjacent to the chromosomes, is important for spindle bipolarity and homolog bi-orientation. These studies suggest that lateral interactions between the chromosomes and microtubules drive homolog bi-orientation, but whether these interactions are kinetochore-based is not clear (Radford, 2015).

There have been few studies directly analyzing kinetochore function in oocyte spindle assembly and chromosome segregation. Assembling a functional spindle requires the initiation of microtubule accumulation around the chromatin, the organization of microtubules into a bipolar structure, and the maturation of the spindle from promoting chromosome alignment to promoting segregation. Whether the kinetochores are required for spindle assembly or the series of regulated and directed movements chromosomes undergo to ensure their proper partitioning into daughter cells is not known. In Drosophila, the chromosomes begin the process within a single compact structure called the karyosome. Within the karyosome, centromeres are clustered prior to nuclear envelope breakdown (NEB). This arrangement, which is established early in prophase and maintained throughout diplotene/diakinesis, is also found in many other cell types. It is possible that the function of centromere clustering is to influence the orientation of the centromeres on the spindle independent of chiasmata. Following NEB, the centromeres separate. In Drosophila oocytes, centromere separation depends on the chromosomal passenger complex (CPC). Whether this movement depends on interactions between chromosomes and microtubules remains to be established (Radford, 2015).

Following centromere separation, homologous centromeres move towards opposite spindle poles. During this time in Drosophila oocytes, the karyosome elongates and achiasmate chromosomes may approach the poles, separating from the main chromosome mass. As prometaphase progresses, the chromosomes once again contract into a round karyosome. These chromosome movements appear analogous to the congression of chromosomes to the metaphase plate that ultimately results in the stable bi-orientation of chromosomes. In mitotic cells, congression depends on lateral interactions between kinetochores and microtubules, and bi-orientation depends on the formation of end-on kinetochore-microtubule attachments. In oocytes, lateral chromosome-microtubule interactions have been suggested to be especially important, but how lateral and end-on kinetochore-microtubule attachments are coordinated to generate homolog bi-orientation has not been studied (Radford, 2015).

To investigate the roles of lateral and end-on kinetochore-microtubule attachments in spindle assembly and prometaphase chromosome movements of acentrosomal oocytes, this study characterized Drosophila oocytes lacking kinetochore components. The KNL1/Mis12/Ndc80 (KMN) complex is at the core of the kinetochore, providing a link between centromeric DNA and microtubules. Both KNL1 and NDC80 bind to microtubules in vitro, but NDC80 is required specifically for end-on kinetochore-microtubule attachments. Therefore, this study examined oocytes lacking either NDC80 to eliminate end-on attachments or the Drosophila homolog of KNL1, SPC105R, to eliminate all kinetochore-microtubule interactions. Oocytes lacking the centromere-associated kinesin motor CENP-E because CENP-E promotes the movement of chromosomes along lateral kinetochore-microtubule attachments in a variety of cell types (Radford, 2015).

This work has identified three distinct functions of kinetochores that lead to the correct orientation of homologs at meiosis I. First, SPC105R is required for the co-orientation of sister centromeres at meiosis I. This is a unique process that fuses sister centromeres, ensuring they attach to microtubules from the same pole at meiosis I. Second, lateral kinetochore-microtubule attachments are sufficient for prometaphase chromosome movements, which may be required for each pair of homologous centromeres to establish connections with microtubules from opposite poles. Third, end-on attachments are dispensable for prometaphase movement but are essential to stabilize homologous chromosome bi-orientation. Surprisingly, it was found that although Drosophila oocytes do not undergo traditional congression of chromosomes to the metaphase plate, CENP-E is required to prevent chromosomes from becoming un-aligned and to promote the correct bi-orientation of homologous chromosomes. It was also shown that the initiation of acentrosomal chromatin-based spindle assembly does not depend on kinetochores, suggesting the presence of important additional interaction sites between chromosomes and microtubules. The stability of the oocyte spindle, however, becomes progressively more dependent on kinetochores as the spindle transitions from prometaphase to metaphase. Overall, this work shows that oocytes integrate several chromosome-microtubule connections to promote spindle formation and the different types of chromosome movements that ensure the proper segregation of homologous chromosomes during meiosis (Radford, 2015).

In acentrosomal oocytes, spindle assembly depends on the chromosomes. How the chromosomes can organize a bipolar spindle that then feeds back and drives processes like bi-orientation of homologous centromeres has been unclear. Previous studies have demonstrated that the central spindle is required for homolog bi-orientation. This study found that several types of functional chromosome-microtubule interactions exist in oocytes, and that each type participates in unique aspects of chromosome orientation and spindle assembly. A model for chromosome-based spindle assembly and chromosome movements in oocytes highlights the multiple and unappreciated roles played by kinetochore proteins such as SPC105R and NDC80, with implications for how homologous chromosomes bi-orient during meiosis I (Radford, 2015).

While the spindle is assembling and becoming organized, the evidence suggests that the chromosomes undergo a series of movements that ultimately result in the bi-orientation of homologous chromosomes. The separation of clustered centromeres is CPC-dependent (Radford, 2012), but not kinetochore-dependent. One possibility is that the CPC-dependent interaction of microtubules with non-kinetochore chromatin drives centromere separation. An alternative is that CPC activity may result in a release of the factors that hold centromeres together in a cluster prior to NEB. A candidate for this factor is condensin, a known target of the CPC, that has been shown to promote the 'unpairing' of chromosomes in the Drosophila germline (Radford, 2015).

Following separation of clustered centromeres, each pair of homologous centromeres bi-orients by separating from each other towards opposite poles. How bi-orientation is established in acentrosomal oocytes is poorly understood. Previous studies in C. elegans and mouse oocytes have suggested a combination of kinetochore-dependent and kinetochore-independent (e.g. involving chromokinesins and chromosome arms) microtubule interactions drive chromosome alignment and segregation. This study found that kinetochores play multiple roles, and the process of chromosome bi-orientation can be broken down into a series of chromosome movements that depend mostly on the kinetochores. First, the centromeres make an attempt at bi-orientation. In Drosophila oocytes, this results in the directed poleward movement of centromeres toward the edge of the karyosome and is accompanied by a stretching of the karyosome. Lateral kinetochore-microtubule attachments mediated by SPC105R are sufficient for this initial attempt at bi-orientation. End-on kinetochore-microtubule attachments via NDC80, however, are essential to maintain the bi-orientation of centromeres. Maintenance of centromere bi-orientation is associated with the stable positioning of the centromeres at the edges facing the poles (Radford, 2015).

The lateral-based chromosome movements required for chromosome orientation are probably mediated by the meiotic central spindle, which have been shown to essential for chromosome segregation. In addition, recent reports in both mitotic and meiotic cells suggest that the initial orientation of chromosomes depends on the formation of a 'prometaphase belt' that likely brings centromeres into the vicinity of the central spindle. Therefore, it is proposed that the initial attempt at bi-orientation occurs during the period when both kinetochores and the central spindle are required for spindle stability. Then, as the oocyte progresses toward metaphase, and the central spindle decreases in importance, this reflects a trend toward the formation of stable end-on kinetochore-microtubule attachments that, in turn, stabilize the bipolar spindle. This model is also corroborated by evidence from mouse oocytes that stable end-on kinetochore-microtubule attachments form after a prolonged prometaphase (Radford, 2015).

The data demonstrate that some chromosome movements, critical for bi-orientation, are dependent on lateral kinetochore-microtubule attachments. The kinetochore-associated kinesin motor CENP-E is thought to be responsible for chromosome movement along lateral kinetochore-microtubule attachments, resulting in chromosome alignment on the metaphase plate. However, because Drosophila meiotic chromosomes are compacted into a karyosome prior to NEB, they do not need to migrate in a plus-end-directed manner to achieve congression and alignment. Instead, centromeres must move toward the poles, perhaps in a minus-end directed manner, to achieve bi-orientation. Interestingly, this study found that CENP-E opposes this minus-end directed movement because in the absence of CENP-E, the karyosome split via lateral kinetochore-microtubule attachments. It is not yet clear what mediates the minus-end-directed movement, but the motors Dynein and NCD (the Drosophila kinesin-14 homolog) or microtubule flux are prime candidates (Radford, 2015).

This study also observed that CMET (CENP-E) is required for the correct bi-orientation of homologous chromosomes. The function proposed in opposing minus-end directed movement may be required for making the correct attachments. As the centromere moves to the edge of the karyosome, CENP-E may not only prevent its separation from the karyosome, but could also force it back towards the opposite pole in cases where the homologs are not bi-oriented. A similar idea has been proposed for CENP-E in mouse oocytes. Alternatively, CENP-E has a second function in tracking microtubule plus-ends and regulating kinetochore-microtubule attachments. In fact, this study found that end-on kinetochore-microtubule stability is affected in the absence of CENP-E. Regulating the stability of microtubule plus-end attachments with kinetochores is critical for establishing correct bi-orientation of homologs. Therefore, both functions of CENP-E could contribute to the correct bi-orientation of centromeres in Drosophila oocytes (Radford, 2015).

Loss of SPC105R has a more severe phenotype than loss of either NDC80 or CENP-E, consistent with a role as a scaffold. It recruits additional microtubule interacting proteins like NDC80 and CENP-E and also recruits checkpoint proteins such as ROD. In analyzing oocytes lacking SPC105R, another class of factors it may recruit was discovered: proteins required for co-orientation of sister centromeres during meiosis I. Co-orientation is a process that fuses the core centromeres and is important to ensure that two sister kinetochores attach to microtubules that are attached to the same spindle pole. Co-orientation could involve a direct linkage between sister kinetochores, as may be the case with budding yeast Monopolin or in maize, where a MIS12-NDC80 linkage may bridge sister kinetochores at meiosis I [56]. In contrast, in fission yeast meiosis I, cohesins are required for co-orientation. Cohesion is stably maintained at the core centromeres during meiosis I but not mitosis, and this depends on the meiosis-specific proteins Moa1 and Rec8. There is also evidence that Rec8 is required for co-orientation in Arabidopsis and this study found that loss of ORD, which is required for meiotic cohesion, also results in a loss of centromere co-orientation. Further studies, however, are necessary to determine if cohesins are required for co-orientation in Drosophila. Indeed, the proteins and mechanism that mediate this process in animals has not been known. Recently, however, the vertebrate protein MEIKIN has been found to provide a similar function to Moa1. Interestingly, both Moa1 and MEIKIN depend on interaction with CENP-C, but do not show sequence homology. Thus, Drosophila may have a Moa1/MEIKIN ortholog that has not yet been identified. In the future, it will be important to identify the proteins recruited by SPC105R and their targets in maintaining centromere co-orientation and how these interact with proteins recruited by CENP-C. The mechanism may involve the known activity of SPC105R in recruiting PP1, because PP1 has been shown to have a role in maintaining cohesion in meiosis I of C. elegans (Radford, 2015).

This study's model for spindle assembly and chromosome orientation raises several important questions for future consideration. The CPC is required for spindle assembly in Drosophila oocytes and the current results highlight the importance of two CPC targets in homolog bi-orientation. One target is central spindle proteins, possibly through the CPC-dependent recruitment of spindle organization factors such as Subito. The CPC is also required for kinetochore assembly, similar to what has been shown in yeast, human cells, and Xenopus and consistent with the finding in human cells that Aurora B promotes recruitment of the KMN complex to CENP-C. It will be important to identify targets of the CPC that drive the initiation of spindle assembly, centromere separation, and bi-orientation. In addition, while this study has found that the CPC is required for kinetochore assembly, it is not known if the CPC promotes error correction by destabilizing kinetochore-microtubule attachments. The CPC may not promote kinetochore-microtubule detachment during meiosis because of the different spatial arrangement of sister centromeres during meiosis I. Indeed, it is not known what is responsible for correcting incorrect attachments at meiosis I or how they are differentiated from correct attachments (Radford, 2015).

In prometaphase, the central spindle and kinetochores contribute to spindle stability. The current data suggests that the kinetochores increase in importance as the oocyte progresses to metaphase, perhaps as a result of the stabilization of end-on kinetochore-microtubule attachments as homologous chromosomes become bi-oriented. However, lateral kinetochore-microtubule interactions demonstrated some resistance to colchicine and allow bivalents to stretch in mouse oocytes. Thus, further studies are necessary to determine if lateral kinetochore-microtubule interactions also confer some stability. The current model also proposes that the transition from prometaphase to metaphase involves a switch from dynamic lateral kinetochore-microtubule interactions to stable end-on kinetochore-microtubule attachments. This transition involves the loss of central spindle microtubules, which occurs regardless of microtubule attachment status. Further studies will be necessary to determine if the prometaphase-to-metaphase transition is developmentally regulated rather than being controlled by the spindle assembly checkpoint. As proposed in mouse oocytes, this may contribute to the propensity for chromosome segregation errors in acentrosomal oocytes by closing the window of opportunity for error correction after key developmental milestones have been passed. Finally, one of the most poorly understood features of meiosis is co-orientation of sister centromeres at meiosis I. What SPC105R interacts with to mediate co-orientation will provide the first insights into the mechanism and regulation of this process in Drosophila (Radford, 2015).

The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. This study employed hydrogen-deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 (Nnf1a and Nnf1b) form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, it is concluded the organization of the Mis12-Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function (Richter, 2016).

Cell division relies on organization of a microtubule (MT) spindle to which replicated chromosomes become attached for equal segregation. Defective MT attachment to kinetochores (KTs) leads to chromosome mis‐segregation and formation of fragmented nuclei after cell division. These events have been proposed to contribute to the genome instability observed in many cancers, indicating that control of MT attachment is a major tumor suppressing process (Morelli, 2016).

The molecular nature of the outer KT, the structure that mediates MT attachment, has been studied extensively. MTs are engaged and stabilized by the Knl1, Mis12, and Ndc80 complexes, together referred to as the KMN network. The KMN network also holds in place the Rod‐Zw10‐Zwilch (RZZ) complex and spindle assembly checkpoint (SAC) proteins, which are important for signaling incomplete attachment. In mammalian cells, MT attachment is further assisted by the KNL1‐interactor ZWINT and by the SKA complex, which associates with curved MT ends at KTs (Morelli, 2016).

In sheer contrast with the extensive molecular knowledge of the outer KT, much less is known about the steps that regulate its assembly. In Drosophila, a widely used metazoan model system for KT studies, part of the Mis12 complex resides at the KT throughout the cell cycle, while the rest of the outer KT is created de novo in early prophase, by stepwise addition of components. The earliest components added to the outer KT in early prophase appears to be Knl1, followed by the Ndc80 complex, both of which are recruited from unknown cellular locales, and SAC components, such as Mad1 and Mad2, which are recruited from nuclear pores (Morelli, 2016).

Unexpectedly, it has been recently found that the Drosophila SNARE protein Snap29 can be isolated from cell extracts together with multiple components of the KMN network. These are the Drosophila Knl1 ortholog Spc105R, three out of four components of the Ndc80 complex (Nuf2 and the Drosophila Spc24 ortholog Kmn2) as well as three of the four subunits of the Mis12 complex (Mis12, Nnf1b, and the Drosophila Nsl1 ortholog Kmn1). SNARE (Soluble NSF Attachment REceptor) proteins (SNAREs) are part of the conserved coiled‐coil machinery that brings membranes in close proximity during trafficking, a prerequisite for most membrane fusion events. The Synaptosomal‐Associated Protein (SNAP) family of SNAREs in metazoans includes Snap25, Snap23, and Snap29, which are composed by two SNARE domains, separated by a linker region. The first two proteins are membrane‐associated and control synaptic transmission and a wide range of non‐neuronal membrane fusion processes, respectively. In contrast, Snap29 only transiently associates with membranes and contains an acidic NPF motif that mediates its association with endocytic factors. Such unconventional features, which are exclusive of Snap29 among the SNARE proteins, predict involvement in a versatile set of membrane trafficking processes, in line with reports in the literature. Consistent with this, Snap29 also controls fusion of autophagosomes with endo‐lysosomes in Drosophila and human cells, together with the SNAREs syntaxin17 (Syx17) and Vamp7 (VAMP8 in human cells) (Morelli, 2016).

Despite involvement of Snap29 in multiple trafficking pathways in interphase, a possible function during cell division has not been explored. This study investigated whether Snap29 localizes and acts at the KT in cells and tissues. The data identify a novel step of KT formation that is conserved and supports tissue formation (Morelli, 2016).

The data uncover an essential and conserved step of KT formation that occurs in prophase and requires Snap29. Such step controls localization of Knl1 (and ZWINT in human cells) to KTs. Snap29 and the RZZ components Rod and Zw10 are known to act in membrane transport between the Golgi apparatus and the ER, and the RZZ complex shares similarity with ER tethering complexes. Strikingly, the autophagosome, which depends on Snap29 for fusion to lysosomes, is formed de novo using Golgi and ER components and engages MTs for dynein‐directed transport to lysosomes, evoking tantalizing similarities between aspects of membrane trafficking and KT formation. Overall, the current data support the possibility that Snap29, Knl1, and the RZZ complex might act at the KT similarly to tethering and fusion complexes existing on membranes, which need to stabilize MTs during trafficking events. Such scenario might imply common ancestry, and underscored also the existence of protozoa that divide with KTs associated with the nuclear membrane that is itself attached to MT fibers (Gómez‐Conde, 2000) (Morelli, 2016).

Ectopic recruitment of Knl1 and possibly RZZ and SKA complex components at sites of SNAP29 Q1 Q2 trapping predicts that the trafficking and KT functions of Snap29 are interconnected, perhaps because of the existence of a common cellular pool of Snap29. A possibility that awaits further investigation is that SNARE domains of Snap29 interact with KT proteins directly. Interestingly, the C‐terminal part of Knl1, Zwint, and Mis12 and Nnf1 all contain multiple coiled‐coil regions. These are placed at the interaction surface between the Mis12, Ndc80, and Knl1 complexes, exactly where Snap29 is seen located by super‐resolution microscopy. In mammalian cells, the Snap29 paralog Snap25 binds Zwint, and Snap29 has been found in Zwint immunoprecipitations. Thus, Snap29 could dock to the Mis12 complex with a SNARE domain and could stabilize interactions with Knl1 and Zwint with the second C‐terminal SNARE domain. Interestingly, no Zwint homolog has been found in Drosophila, suggesting that in flies Snap29 could substitute for Zwint. The ability of KNL1 to interact with a SNAP29 that cannot be released from SNARE complex, both suggest that the interaction of KNL1 with SNAP29 might occur on the side of the SNARE domain that is not occupied by a syntaxin or a Vamp. These data also suggest that SNAP29 could act on Knl1 also prior to nuclear entry and KT localization (Morelli, 2016).

The evidence in vivo indicates that SNAP29 function could support tissue development by ensuring faithful chromosome segregation and that such activity is crucial in cells that become resistant to apoptosis. Based on this, it is predicted that loss of SNAP29 could be selected in cancers with highly unstable genomes. In addition, rare congenital syndromes such as CEDNIK, Roberts syndrome, and primary microcephaly (MCPH) are caused by mutations in Snap29 and other genes encoding proteins that regulate mitosis, respectively, overall suggesting that ability to cope with defective mitotic cells is major process for tissue development and homeostasis (Morelli, 2016).

Centromeres are the chromosomal sites of assembly for kinetochores, the protein complexes that attach to spindle fibers and mediate separation of chromosomes to daughter cells in mitosis and meiosis. In most multicellular organisms, centromeres comprise a single specific family of tandem repeats, often 100-400 bp in length, found on every chromosome, typically in one location within heterochromatin. Drosophila melanogaster is unusual in that the heterochromatin contains many families of mostly short (5-12 bp) tandem repeats, none of which appear to be present at all centromeres, and none of which are found only at centromeres. Although centromere sequences from a minichromosome have been identified and candidate centromere sequences have been proposed, the DNA sequences at native Drosophila centromeres remain unknown. This study use native chromatin immunoprecipitation to identify the centromeric sequences bound by the foundational kinetochore protein cenH3, known in vertebrates as CENP-A. In D. melanogaster, these sequences include a few families of 5-bp and 10-bp repeats, but in closely related D. simulans, more complex repeats comprise the centromeres. The results suggest that a recent expansion of short repeats has replaced more complex centromeric repeats in D. melanogaster (Talbert, 2018).

The kinetochore is a complex of proteins, broadly conserved from yeast to man, that resides at the centromere and is essential for chromosome segregation in dividing cells. There are no known functions of the core complex outside of the centromere. This study shows that the proteins of the kinetochore have an essential post-mitotic function in neurodevelopment. At the embryonic neuromuscular junction of Drosophila melanogaster, mutation or knockdown of many kinetochore components cause neurites to overgrow and prevent formation of normal synaptic boutons. Kinetochore proteins were detected in synapses and axons in Drosophila. In post-mitotic cultured hippocampal neurons, knockdown of mis12 increased the filopodia-like protrusions in this region. It is concluded that the proteins of the kinetochore are repurposed to sculpt developing synapses and dendrites and thereby contribute to the correct development of neuronal circuits in both invertebrates and mammals (Zhao, 2019).